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smear for digestion


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#1 shyann

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Posted 10 November 2009 - 04:12 AM

I am digesting pET32b vector using BamHI and HIndIII. I've tried 6-7 times and I got smear. I've did a reaction with plasmid+dH20+buffer and i got a smear. Is it contamination? Contaminated with nuclease? But I am extracting the vector from E.coli, should be no problem with it. My collegue did the same thing as mine,but she got a nice band after 2 hrs digestion. I've tried 2hrs,1 hrs and even half an hour, but all i got is a smear. Can somebody tell me what is the problem?

#2 stardust

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Posted 10 November 2009 - 04:14 AM

Hi there!

How are you isolation the plasmid from E. coli? With a kit or selfmade miniprep?

Maybe you are vortexing at the wrong steps and your samples conatin E. coli genomic DNA? Did you run a sample of your plasmid prep without treatment?

Stardust

#3 shyann

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Posted 10 November 2009 - 05:53 AM

Hi there!

How are you isolation the plasmid from E. coli? With a kit or selfmade miniprep?

Maybe you are vortexing at the wrong steps and your samples conatin E. coli genomic DNA? Did you run a sample of your plasmid prep without treatment?

Stardust


I am using promega extraction kit. I've run the plasmid without treatment. I got 3 band, which mean 3 conformation for plasmid,right?
And I am adding 0.2uL of enzyme to the reaction, I think it is not over digestion. I didn't vortexing it except while resuspending the cells. Other steps like cell lysis etc. I am just inverted it 4 times. Is it my technical problem or what?

#4 Vini

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Posted 10 November 2009 - 07:01 AM

Hi there!

How are you isolation the plasmid from E. coli? With a kit or selfmade miniprep?

Maybe you are vortexing at the wrong steps and your samples conatin E. coli genomic DNA? Did you run a sample of your plasmid prep without treatment?

Stardust


I am using promega extraction kit. I've run the plasmid without treatment. I got 3 band, which mean 3 conformation for plasmid,right?
And I am adding 0.2uL of enzyme to the reaction, I think it is not over digestion. I didn't vortexing it except while resuspending the cells. Other steps like cell lysis etc. I am just inverted it 4 times. Is it my technical problem or what?


seems like your DNA is getting degraded while setting up the R.E. reaction. For one, make sure of that. Another thing could be that the enzymes are showing star activity. Are you sure that you are using a compatible buffer for both enzymes?

#5 shyann

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Posted 10 November 2009 - 07:43 AM

Hi there!

How are you isolation the plasmid from E. coli? With a kit or selfmade miniprep?

Maybe you are vortexing at the wrong steps and your samples conatin E. coli genomic DNA? Did you run a sample of your plasmid prep without treatment?

Stardust


I am using promega extraction kit. I've run the plasmid without treatment. I got 3 band, which mean 3 conformation for plasmid,right?
And I am adding 0.2uL of enzyme to the reaction, I think it is not over digestion. I didn't vortexing it except while resuspending the cells. Other steps like cell lysis etc. I am just inverted it 4 times. Is it my technical problem or what?


seems like your DNA is getting degraded while setting up the R.E. reaction. For one, make sure of that. Another thing could be that the enzymes are showing star activity. Are you sure that you are using a compatible buffer for both enzymes?


emmm...I don't think is because of star activity. I am using a compatible buffer for both enzyme as they are having the same optimal buffer conditions. I am thinking that why the reaction that doesn't contain enzyme will get a smear too...

#6 Vini

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Posted 10 November 2009 - 08:09 AM

Hi there!

How are you isolation the plasmid from E. coli? With a kit or selfmade miniprep?

Maybe you are vortexing at the wrong steps and your samples conatin E. coli genomic DNA? Did you run a sample of your plasmid prep without treatment?

Stardust


I am using promega extraction kit. I've run the plasmid without treatment. I got 3 band, which mean 3 conformation for plasmid,right?
And I am adding 0.2uL of enzyme to the reaction, I think it is not over digestion. I didn't vortexing it except while resuspending the cells. Other steps like cell lysis etc. I am just inverted it 4 times. Is it my technical problem or what?


seems like your DNA is getting degraded while setting up the R.E. reaction. For one, make sure of that. Another thing could be that the enzymes are showing star activity. Are you sure that you are using a compatible buffer for both enzymes?


emmm...I don't think is because of star activity. I am using a compatible buffer for both enzyme as they are having the same optimal buffer conditions. I am thinking that why the reaction that doesn't contain enzyme will get a smear too...



oh yeah, i missed the latter part in your post....problem with dH2O?? tried using another aliquot??? what about the buffer....maybe that has got contaminated???

#7 shyann

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Posted 10 November 2009 - 06:02 PM

I've tried to use different buffer,but smear too. For the distill water...i aliqout into 1.5uL microcentrifuge tube after autoclave. Everytime doing digestion, i am using a new one. So,is there any problem with the distill water? Or should i use nuclease-free water?

#8 phage434

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Posted 10 November 2009 - 06:10 PM

There is no way to tell what is happening without much more information about your reaction. Tell us precisely how you are preparing your DNA, what concentration it is,
precisely what volumes and amounts of enzymes are used, and how much you are running on a gel. We can't guess effectively.

#9 shyann

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Posted 10 November 2009 - 09:15 PM

There is no way to tell what is happening without much more information about your reaction. Tell us precisely how you are preparing your DNA, what concentration it is,
precisely what volumes and amounts of enzymes are used, and how much you are running on a gel. We can't guess effectively.


I'm extracting plasmid from 10mL culture by using extraction kit. Use 25uL of steriled distill water to elute it, and its concentration is around 300ng/uL. I then digest 5uL of it by using 0.5uL of enzyme (10U/uL) and incubate for 3 hrs. Because it appear as smear, i then try to use 0.2uL of enzyme and incubate for 3hrs,2 hrs and 1 hr. All appear as smear.
My digestion reaction is 20uL and i put all to run on the gel.

#10 phage434

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Posted 11 November 2009 - 05:56 AM

That's some information, but not enough. I would replace the water for elution with TE. 10 ml of culture sounds as if you may be overloading the column -- try 2 ml instead. What volume are you doing the restriction digest in? Too much DNA in low volumes can lead to problems. Aim for 100 ng/ul DNA concentration from your prep, and cut 10 ul (1 ug) in at least 50 ul of restriction digest. I'm guessing the problem is overload of your column and extraction of genomic DNA during your "plasmid" prep. Also check to make sure that you have added RNAse to the initial resuspension buffer -- the smear could be RNA coming through the miniprep. What enzyme are you using? Buffer? Amounts? Do you add BSA? We need to know it all.

#11 shyann

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Posted 11 November 2009 - 08:02 AM

That's some information, but not enough. I would replace the water for elution with TE. 10 ml of culture sounds as if you may be overloading the column -- try 2 ml instead. What volume are you doing the restriction digest in? Too much DNA in low volumes can lead to problems. Aim for 100 ng/ul DNA concentration from your prep, and cut 10 ul (1 ug) in at least 50 ul of restriction digest. I'm guessing the problem is overload of your column and extraction of genomic DNA during your "plasmid" prep. Also check to make sure that you have added RNAse to the initial resuspension buffer -- the smear could be RNA coming through the miniprep. What enzyme are you using? Buffer? Amounts? Do you add BSA? We need to know it all.


The plasmid that I extract is low-copy number. If use 2mL will too little or not?
I am setting up 20uL of digestion reaction with 2uL buffer E, 0.2uL BSA, 0.5uL HindIII, 0.5uL BamHI, 5.0uL plasmid (~300ng/uL), dH2O.
But after i got a smear for it,i reduce the enzyme volume to 0.2uL.
I didn't use RNAse...I just follow the protocol of extraction kit... :( hmm...i think i will try to add RNAse next time...thanks ya...
Beside, still got any problem with my digestion?

#12 phage434

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Posted 11 November 2009 - 02:11 PM

I would immediately retry the digestion at lower concentration. You are digesting 1.5 ug of DNA in 20 ul, which is almost 4 times the recommended concentration of 1 ug in 50 ul. It's difficult to pipet accurately 0.5 ul of enzyme, so it is easy to approach too high levels of glycerol in a low volume reaction. Enzyme inhibitors may be present in your DNA preparation, which can inhibit cutting. With a higher volume, you dilute those inhibitors substantially (4x dilution in your reaction setup, vs. 16x dilution in a 1 ug, 50 ul reaction).




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